The aim of the proposed studies is to gain insight into the biosynthesis, assembly and organization of cytoskeletal components of cultured human fibroblasts during senescence. An unusual abundance of filamentous structures in the cytoplasm is one of the outstanding phenotypic characteristics found in aging cells, whose size has become enlarged and whose proliferation has either slowed down or ceased. In the past we and others have demonstrated that cell morphology is largely dependent upon the state of assembly and organization of three cytoskeletal structures: microtubules, 10 nm filaments and microfilaments. Using cultured fibroblasts derived from human donors of varying ages as a model system, we plan to investigate: (1) the precise structural features of the distribution and organization of cytoskeleton in senescent cells; (2) the state of cytoskeletal biosynthesis in slowly proliferating fibroblasts; (3) the interaction between cytoskeletal proteins and the plasma membrane that may govern the assembly and organization of the cytoskeleton; and (4) the alterations in the equilibrium for the assembly and disassembly of cytoskeletal proteins during senescence. Information will be obtained through morphological studies using light and electron microscopy and quantitative analysis using various biochemical techniques. In addition, the functional expressions of the cytoskeleton in senescent cells, such as locomotion, intracellular movements, and cell-surface movement will also be investigated. We will also attempt to reinitiate cell proliferation in senescent cultures by cell fusion between slowly proliferating aging cells and rapidly growing tumor virus-transformed cells. Information obtained from this project will at least partially answer questions such as why and how the cytoskeletal system proceeds in the direction of increased assembly and organization during senescence. Our long term goal is to analyze how changes in the biosynthesis and organization of cytoskeleton are coordinated with the state of other cellular components into a mechanism for restricting cell proliferation during aging.